Comparative proteomic analysis of high‐ and low‐fertile buffalo bull spermatozoa for identification of fertility‐associated proteins

The present study identified few potential proteins in the spermatozoa of buffalo bulls that can be used as an aid in fertility determination through comparative proteomics. The sperm proteome of high‐fertile buffalo bulls was compared with that of low‐fertile buffalo bulls using two‐dimensional difference gel electrophoresis (2D‐DIGE), and the differentially expressed proteins were identified through mass spectrometric method. The protein interaction network and the functional bioinformatics analysis of differentially expressed proteins were also carried out. In the spermatozoa of high‐fertile bulls, 10 proteins were found overexpressed and 15 proteins were underexpressed at the level of twofold or more (p ≤ 0.05). The proteins overexpressed in high‐fertile spermatozoa were PDZD8, GTF2F2, ZNF397, KIZ, LOH12CR1, ACRBP, PRSS37, CYP11B2, F13A1 and SPO11, whereas those overexpressed in low‐fertile spermatozoa were MT1A, ATP5F1, CS, TCRB, PRODH2, HARS, IDH3A, SRPK3, Uncharacterized protein C9orf9 homolog isoform X4, TUBB2B, GPR4, PMP2, CTSL1, TPPP2 and EGFL6. The differential expression ranged from 2.0‐ to 6.1‐fold between the two groups, where CYP11B2 was high abundant in high‐fertile spermatozoa and MT1A was highly abundant in low‐fertile spermatozoa. Most of the proteins overexpressed in low‐fertile spermatozoa were related to energy metabolism and capacitation factors, pointing out the possible role of pre‐mature capacitation and cryo‐damages in reducing the fertility of cryopreserved buffalo spermatozoa.     

Partial cDNA sequence of a relaxin‐like factor (RLF) receptor,LGR8 and possible existence of the RLF ligand‐receptor system in goat testes

Relaxin‐like factor (RLF), also known as insulin‐like factor 3 (INSL3), is produced by testicular Leydig cells, but its specific receptor LGR8 (leucine‐rich repeat family of G‐protein‐coupled receptor 8) has not been identified in goats. This study aimed to identify complementary DNA (cDNA) sequences of goat LGR8, and characterize the expression of both RLF and LGR8 in goat testes by RT‐PCR and immunohistochemistry. Testes were collected from immature (3‐month‐old) and mature (24‐month‐old) Saanen goats, and partial cDNA sequences of the goat homologue of human LGR8 were identified. The sequence encoded a reduced peptide sequence of 167 amino acids, which corresponded to transmembrane regions 2 through 5, followed by the beginning of intracellular loop 3 of human LGR8. Expression of both LGR8 and RLF genes was drastically increased in mature testes compared with immature ones. Although RLF protein was restricted to Leydig cells, LGR8 protein was detected in both Leydig cells and seminiferous epithelial cells (possibly germ cells and Sertoli cells). These results reveal a possible existence of the RLF‐LGR8 ligand‐receptor system within the goat testis, suggesting that RLF may play a role in testicular function through LGR8 on Leydig cells and seminiferous epithelial cells in an autocrine and/or paracrine manner.     

Alkaline and Acid Phosphatase, β‐Glucuronidase and Electrolyte Levels in Fractionated Stallion Ejaculates

Seminal plasma (SP) is a mixture of contents from the testes, epididymides and accessory sex glands. The sperm concentration is highest in the first few jets, or fractions, of the ejaculate, and the composition of SP varies between these fractions because accessory gland secretions are released in a specific order. The aim of this study was to compare the levels of Na, Cl, K, Mg, Ca, inorganic phosphate (Pi) and the enzymes alkaline phosphatase (AP), acid phosphatase (ACP) and β‐glucuronidase (BG) in the different fractions of the ejaculate and in different stallions. All semen collections were done using a computer‐controlled phantom that collects the ejaculatory jets separately in five cups. The cups with the highest (HIGH) and the lowest (LOW) sperm concentration were analysed. In Trial I, semen was collected from three reproductively normal stallions. In Trial II, ejaculates of two reproductively normal stallions were compared to those of two subfertile stallions. In Trial III, semen was collected from seven stallions with varying reproductive history. The sperm‐rich fractions contained the highest levels of AP, ACP, BG and inorganic phosphate, and the values were positively correlated to the sperm concentration. Significant differences between the subfertile and the fertile stallions pairs in HIGH : LOW ratios were found in Pi and Cl concentrations. The highest concentrations of Ca and Mg were found in the last fractions with low sperm concentrations, with no significant differences between the fertile and the subfertile stallion pairs. The concentrations of K, Na and Cl were similar in HIGH and LOW fractions and in whole ejaculate samples. Pre‐sperm fluid contained the highest concentrations of Na and Cl. Some of the possible variation in storage tolerance between ejaculates and ejaculatory fractions could perhaps be explained by differences in the composition of SP.     

Changes in Semen Quality in Estonian Holstein AI Bulls at 3, 5 and 7 years of Age

The predictability of semen quality of mature sires from measurements at an early age is not well established. The aim of the present study was to determine age-dependent changes in the quality of bull semen from six Estonian Holstein (EHF) bulls, processed when the sires were 3, 5 and 7 years old. Fertility data such as 60-day non-return to oestrus rates (60d-NRRs) were available for 3-year-old bulls. From each batch, semen straws were analysed immediately after thawing [i.e. post-thaw (PT)] (controls) and after a swim-up (SU) procedure. The analyses comprised subjective and computerized measurements of sperm motility using computer-assisted sperm analysis (CASA) as well as estimations of sperm concentration, morphology and membrane integrity. There was a significant (p < 0.05) increase in the percentage of sperm motility (SU), membrane integrity (PT, SU) and normal tail and acrosome morphology (SU) with an increase in the age of the sires. The percentage of total motile spermatozoa PT measured by CASA correlated between 3- and 7-, and between 5- and 7-year-old bulls (p < 0.05). In addition, the proportion of head abnormalities tended to correlate between all three age groups both PT and after SU (p < 0.1). The sperm parameters correlating with fertility were average path velocity (VAP) (p < 0.001), total motility as measured by CASA (p < 0.01), linearly motile spermatozoa (p < 0.05) and CASA-assessed numbers of motile spermatozoa (p < 0.05), all after SU selection. The results showed that overall semen quality examined at 3 years of age is related to the semen parameters later in bulls' life. Moreover, CASA-assessed motility after SU seems to be a reliable marker for semen quality assessment as it shows correlation not only between the ages, but also to field fertility.     

Effect of Short‐Term Scrotal Hyperthermia on Spermatological Parameters,Testicular Blood Flow and Gonadal Tissue in Dogs

The objective was to assess the effect of a short‐term scrotal hyperthermia in dogs on quantitative and qualitative ejaculate parameters, testicular blood flow and testicular and epididymal histology. After a control period, the scrotum of seven normospermic adult beagle dogs was insulated with a self‐made suspensory for 48 h. Nine weeks later, two animals were castrated, while in five animals, scrotal hyperthermia was repeated. Dogs were castrated either 10 or 40 days thereafter. In each phase of scrotal insulation, average scrotal surface temperature increased by 3.0°C. Semen was collected twice weekly throughout the experiment. Total sperm count did not change after the first hyperthermia, but it slightly decreased after the second (p < 0.05). Profiles of sperm morphology and velocity parameters (CASA) rather indicated subtle physiological variations in sperm quality than effects of a local heat stress. Chromatin stability of ejaculated spermatozoa as indicated by SCSA remained constant throughout the experiment. Perfusion characteristics of the gonads, that is, systolic peak velocity, pulsatility and resistance index at the marginal location of the testicular artery, did not change due to hyperthermia (p > 0.05). Histological examination of excised testes and epididymides for apoptotic (TUNEL and activated caspase‐3) and proliferating cells (Ki‐67 antigen) indicated only marginal effects of scrotal insulation on tissue morphology. In conclusion, a mild short‐term scrotal hyperthermia in dogs does not cause substantial changes in sperm quantity and quality. In contrast to other species, canine testes and epididymides may have a higher competence to compensate such thermal stress.     

Persistence of undifferentiated spermatogonia in aged Japanese Black cattle

Aging is a major risk factor for spermatogenesis deterioration. However, the influence of age on spermatogenic stem cells and their progenitors in bulls is largely unknown. Here, we report age-related changes in undifferentiated and differentiating spermatogonia in Japanese Black cattle with nearly constant sperm output, by using spermatogonial markers. The numbers of differentiating spermatogonia and more differentiated spermatogenic cells were significantly decreased in aged bovine testes compared with those in young testes. In contrast, the number of undifferentiated spermatogonia was maintained, and their proliferative activity did not differ significantly between young and aged bovine testes. Although severe calcification was only observed to a small extent in aged testes, fewer Sertoli cells and interstitial fibrosis were observed in noncalcified testicular regions. These results suggest that, even in old bulls with nearly constant sperm output, testicular spermatogenic activity declined whereas undifferentiated spermatogonia numbers were maintained. Thus, we propose that undifferentiated spermatogonia may be resistant to age-related changes in bovine testes. Because undifferentiated spermatogonia may contain stem cell activity, our findings highlight the potential utility of undifferentiated spermatogonia as an agricultural resource to produce spermatozoa beyond the natural bovine lifetime through transplantation and in vitro spermatogenesis in future animal production.     

Estimation of endogenous levels of osteopontin,total antioxidant capacity and malondialdehyde in seminal plasma: Application for fertility assessment in buffalo (Bubalus bubalis) bulls

This study was attempted to identify subfertile bulls by quantifying the endogenous levels of osteopontin (OPN), total antioxidant capacity (TAC) and malondialdehyde (MDA) in seminal plasma of buffalo bulls. On the basis of conception rate, buffalo bulls were classified into two groups: high‐fertile (conception rate >50%) and subfertile bulls (conception rate <40%). A total of 100 ejaculates (10 ejaculates from each bull) were collected through artificial vagina method. The concentration of OPN, TAC and catalase (CAT) of high‐fertile bulls was found to be higher (p < .05) than that of subfertile bulls. Further, MDA level in seminal plasma was found to be lower (p < .05) in high‐fertile bulls compared with subfertile bulls. The fertility status had no effect on the superoxide dismutase (SOD) concentration in seminal plasma of both the groups. The levels of OPN (r = .678, p = 0.013) and TAC (r = .648, p = .042) were found to be positively correlated with bull fertility and the level of MDA (r = ?.718, p = .019) was found to be negatively correlated with bull fertility. However, the fertility of bulls was not found to be significantly correlated with SOD, CAT and sperm motility. In conclusion, seminal OPN, TAC and MDA tended to be more realistic in identification of subfertile bulls from breeding herds.     

Morphological and Ultrastructural Studies of Moose Spermatozoa

Epididymal spermatozoa from moose were studied in phase contrast, light interference and electron microscope. Some samples taken from cauda were diluted and frozen in liquid N₂. The motility of the sperms after thawing was good.The concentration of spermatozoa in cauda was calculated to 10 × 10⁶ cells per µl.Morphologically the spermatozoa of moose were found to be quite similar to those collected from bulls. The length of the sperm head was found to be approx. 8.8 µ and the average maximal width 5.2 µ. The average length of the tail was 54.7 µ and the entire length of the spermatozoon varied from 60 to 64 µ. Compared with sperm cells from bulls the moose spermatozoa appeared to have a somewhat shorter and broader head and a slightly shorter tail.The migration of the cytoplasmatic droplets, which was found to be completed in caput, seemed to follow the same pattern as in bulls and boars. As found in these species there was also in the moose a higher frequency of secondary abnormalities in the spermatozoa from cauda than in those from the other parts of epididymis.Studies of the fine structure of the moose spermatozoa seemed to indicate that these are of the same type as the spermatozoa of bulls, rams and boars. In sagittal sections the sperm head was thin, but in contrast to the sperm cells of the species mentioned above no typical waist-like narrowing in the equatorial region was found. The equatorial segment also seemed to be less arched than in the spermatozoa from bulls, rams and boars. Otherwise, no principal difference was found between ultrastructure of the moose spermatozoa and that of the spermatozoa collected from domestic species.     

Flow cytometry evaluation of testicular and sperm cells obtained from bulls implanted with zeranol

Flow cytometry of testicular and sperm cells was used to evaluate effects of pre-weaning zeranol implants on spermatogenesis. Forty five Angus-Simmental bulls were randomly assigned to three treatment groups of 15 bulls each: no implant, one implant at 30 d of age and two implants, one at 30 and the second at 120 d of age. Prior to slaughter at approximately 15 mo, semen was collected from 30 bulls, 10 of each group. Following slaughter, testes were weighed, and testicular biopsies and vas deferens sperm obtained from the same 30 bulls. Testicular and sperm cells were stained with acridine orange and measured by flow cytometry. Proportions of testicular haploid, diploid and tetraploid cells were determined by relative amounts of green (DNA) and red (RNA) fluorescence. Treatment of sperm at low pH prior to acridine orange staining potentially induces partial denaturation of DNA, detectable by the metachromatic shift from green (native DNA) to red (single-stranded DNA) fluorescence. The effect of this shift was quantified by alpha-t [alpha t = red/red + green) fluorescence]. Nonimplanted bulls had heavier (P less than .01) testicular weights than treated bulls. The proportion of haploid cells was greater (P less than .02) and diploid cells less (P less than .03) in testes of nonimplanted bulls. Sperm from implanted bulls had altered chromatin structure, indicated by higher (P less than .05) alpha t values. Flow cytometry is an effective means for detecting changes in testicular cell subpopulations and chromatin structure of sperm.     

Effects of Melatonin Implants During Non‐Breeding Season on Sperm Motility and Reproductive Parameters in Rasa Aragonesa Rams

The effect of melatonin implants administered during non‐breeding season in Rasa Aragonesa rams on sperm motility parameters and other reproductive traits was assessed. In a first experiment, two Rasa Aragonesa rams were implanted (with melatonin group M), remaining other two males as control group (C). Semen of each group was collected from 1 May to 23 June, twice or three times a week, and motility parameters were assessed using a computer‐assisted sperm analysis system. Melatonin increased the percentage of progressive motile spermatozoa, particularly during 46–75 days after melatonin implantation (p < 0.01). In experiment 2, M and C in vitro fertilization ability had been determined by zona‐pellucida binding assays, using spermatozoa from experiment 1, obtained 60–70 days after melatonin was implanted. A significantly higher number of spermatozoa attached per oocyte was observed in frozen‐thawed immature ovine oocytes incubated with sperm from M animals than in those incubated with sperm from the C group (p < 0.01). Finally, a field assay (experiment 3) was performed. In this case, five Rasa Aragonesa rams were implanted with melatonin and three remained as control group. Sperm doses from those animals were used for artificial insemination of 2608 Rasa Aragonesa ewes from 39 different farms at non‐breeding season. Fertility, litter size and fecundity were studied. Semen from melatonin implanted rams seemed to increase both fertility and fecundity in ewes inseminated with spermatozoa obtained 46–60 days after implantation (p < 0.1). Thus, melatonin treatment in rams during non‐breeding season modifies sperm motility parameters and seems to improve the fertilization parameters obtained.     

Pathophysiology of small testes in beef bulls: relationship between scrotal circumference, histopathologic features of testes and epididymides, seminal characteristics, and endocrine profiles

Tissue sections from testes and epididymides obtained from 17 young beef bulls with scrotal circumference (SC) between 27 and 40.5 cm were studied to determine whether small testes were a manifestation of lesions or a result of less, but otherwise normal, seminiferous epithelium. The SC correlated negatively with the estimates of germinal epithelial loss and positively with seminiferous epithelial area. Four bulls with SC less than 30 cm had severe lesions in their testes. Hypoplastic tubules were characterized by Sertoli's cells only with no evidence of germinal cells. Loss of germinal cells, leaving vacuolated epithelium and atrophy, were observed in degenerated tubules. Hyperplasia of Leydig's cells was observed in the vicinity of Sertoli's cell-only tubules, resulting either from degeneration or hypoplasia, and atrophy of Leydig's cells was associated with tubules devoid of Sertoli's cells. These findings indicated that Sertoli's cells may produce a factor(s) required for maintenance and regulation of Leydig's cell function. Epididymal epithelium, especially in the head, had regressed in bulls with hypoplastic and degenerative changes in their testes. Decreased sperm concentration and motility and an increased frequency of morphologic defects were observed in the 4 bulls with testicular lesions and regressed epididymal epithelium. Blood plasma profiles of cortisol, follicle-stimulating hormone, luteinizing hormone, and testosterone were determined in the 4 bulls with SC less than 30 cm and 10 of the 13 bulls with SC greater than 30 cm. There were no statistically significant (P greater than 0.1) differences in the responses to exogenous gonadotropin-releasing hormone or base-line patterns of blood plasma follicle-stimulating hormone and luteinizing hormone between the 2 groups. However, in the bulls with SC less than 30 cm, the mean concentration of testosterone was lower, whether spontaneous (P less than 0.05) or exogenous gonadotropin-releasing hormone induced (P less than 0.1). The fact that these bulls were not deficient in gonadotropins indicated that Leydig's cell function was impaired by local factors, either the factors that caused the tubular damage or those consequent to the tubular damage.     

Germ cell weakness as a cause of testicular hypoplasia in bulls.

Sporadic cases of testicular hypoplasia were earlier found in bulls of the Swedish Red and White breed. An accumulation of cases have occurred since 1970 in sons of 2 outstanding progenytested bull sires, 2 F and 27 U, which had a common father, 545 B. The history and clinical examination of affected bulls varied. Some had azoospermia and very small testes at a young age, while others could be normal in all respect when they were young but had a short reproductive life and had to be culled at about 3 years of age. Most of the affected bulls were between these 2 extremes. The histologic examination showed principally different degrees of testicular degeneration. There were always some germ cells left in all affected seminiferous tubules indicating that there was not a lack of germ cells causing the hypoplasia. Germ cell weakness is obviously a hereditary condition. The sires 545 B, 2 F and 27 U had a relatively low fertility. In their pedigree were several bulls known to have had a low fertility. No sons of 2 F and only a few sons of 27 U were used for A.I. services and at present only few cases of testicular hypoplasia are seen.     

Molecular expression of caprine estrogen receptor gene 1 in reproductive and non‐reproductive tissues

During the last decades, physiological effects of oestrogens have been increasingly explored by scientists and biotechnologists. Estrogens exert a wide range of effects on a large variety of cell types. Oestrogen and its receptors are essential for sexual development and reproduction. Estrogen receptor alpha is a nuclear receptor activated by the hormone oestrogen. In male, ERα is encoded by the gene estrogen receptor gene 1 (ESR1), responsible for better fertility. The ESR1 is involved in the reabsorption of luminal fluid during the transit of spermatozoa from the testis to the head of the epididymis which is important for their survival and maturation during epididymal storage. The absence of ESR1 leads to reduced epididymal sperm content, reduced sperm motility and fertilizing ability. Therefore, this is a good startby to study the expression pattern of estrogen receptor 1 gene in high‐fertile (G1) and low‐fertile (G2) bucks of Jamunapari and Barbari breeds identified on the basis of seminal quality traits and fertility trials. RNA was extracted from the tissues by TRIzol method. The identification and expression pattern of caprine ESR1 gene was analysed by real‐time PCR (Roche LC‐480). Our work shows that the relative quantification by RT‐PCR indicates more fold in head of epididymis as compared to spleen of caprine ESR1 gene. Furthermore, the RT‐PCR indicated that fertile bucks of Jamunapari breed have more fold value as compared to Barbari breed in respect of reproductive organ.     

Seminiferous epithelium cycle staging based on the development of the acrosome in ram testis

Testicular histopathology is considered the most sensitive and reliable method to detect the effects of chemicals on sperm production. To carry out a sensitive examination of testicular histopathology and interpret the changes require knowledge of spermatogenic stages. Spermatogenic staging based on acrosome development during spermiogenesis is conventionally performed in animal species routinely used for research and toxicity testing. In contrast, small ruminants, such as sheep and goats, are rarely used as animal models to evaluate toxicity in male reproductive organs. To the best of our knowledge, a comparable spermatogenic staging system in rams has not yet been fully characterised. Hence, this study aimed to adapt the existing spermatogenic staging based on acrosome development in bull testes to fit the seminiferous epithelium cycle of ram testes. The results show that spermatogenic staging based on acrosome development in bull testes can, with slight modifications, be efficiently used for the staging of ram testes.     

Effect of Bovine Sperm‐Bound Antisperm Antibodies on Oviductal Binding Index

This study was designed to test the hypothesis that sperm‐bound IgG and IgA decrease binding of bull spermatozoa to oviductal epithelial cells in vitro. Three ejaculates were cryopreserved from each of four antisperm antibody (ASA)‐negative satisfactory breeder bulls. Bulls were then immunized with autologous spermatozoa, and three ASA‐positive ejaculates were cryopreserved from each bull post‐immunization. First, microscopy methods were compared to select the most appropriate assay for evaluation of oviductal binding index (BI). The BI did not differ when the evaluation was performed under fluorescence microscopy (131.1 sperm/mm²; 62.5–251.1 sperm/mm²), phase‐contrast microscopy (160.5 sperm/mm²; 56.8–397.4 mm²) or their combination (116.4 sperm/mm²; 56.8–249.6 sperm/mm²) (Median; IQR). The combination of microscopy methods was selected as it allowed better visualization of cells. Then, BI was compared between ASA‐negative and ASA‐positive ejaculates, and the association between BI and ASA binding was evaluated. The BI was less in ASA‐positive (114.9; 0 to 201.8 sperm/0.1 mm²) than ASA‐negative samples (218.9; 24.7 to 276.8 sperm/0.1 mm²) (P = 0.0002). This reduction in BI was significant in three of the four bulls. Regression analysis identified a negative association between BI and the percentage of IgG‐bound (p = 0.013) but not IgA‐bound spermatozoa. In conclusion, sperm‐bound IgG decreased the ability of bovine spermatozoa to bind to oviductal epithelial cells in vitro.     

Expression of Matrix Metalloproteinases (MMP‐2, MMP‐9) and their Inhibitors (TIMP‐1, TIMP‐2) in Canine Testis,Epididymis and Semen

This study aims to investigate the role of matrix metalloproteinases (MMPs) in determining semen quality and to evaluate the expression and cellular localization of MMP‐2, MMP‐9, tissue inhibitor of metalloproteinase‐1 (TIMP‐1) and TIMP‐2 in the testes, epididymis and ejaculated spermatozoa. Gelatinase activities between normal (n = 21) and abnormal (n = 25) semen samples showed a significant, sixfold increase in proMMP‐2 and MMP‐2 activity in high than low sperm concentration samples (p < 0.001). ProMMP‐9 and MMP‐9 levels were significantly elevated in samples with low sperm counts compared to those with high sperm density (p < 0.001). High levels of proMMP‐2 and MMP‐2 were associated with high sperm motility (≥70%, p < 0.001). Sperm‐rich fraction showed significantly (eight‐fold) higher proMMP‐9 enzymatic activity compared with prostatic fraction. The mRNA expressions of MMP‐2, MMP‐9, TIMP‐1 and TIMP‐2 were confirmed in testicular and epididymal tissues. Immunohistochemical staining illustrated the MMP‐2‐specific strong immunoreactivity in the head of mature spermatids during spermatogenesis, whereas MMP‐9, TIMP‐1 and TIMP‐2 were absent in these cells. Matrix metalloproteinase‐9 immunoreactivity was observed in the spermatocyte and round spermatid, whereas TIMP‐1 was only exhibited in the residual bodies. Immunolabeling of epididymal and ejaculated sperm demonstrated MMP‐2 localization along acrosomal region of sperm, while MMP‐9, TIMP‐1 and TIMP‐2 localization was merely limited to the flagella. In conclusion, spermatozoa initially acquire MMP‐2 during their formation at testicular level, and the presence of this protein persists through the epididymal transit and up to ejaculate. The enzymatic activity of MMP‐2 and MMP‐9 may serve as an alternative biomarker in determining semen quality.     

Genomewide analysis of bull sperm quality and fertility traits

Because the priority of AI industry is to identify subfertile bulls, a predictive model that allowed for the prediction of 91% bulls of low fertility was implemented based on seminological (motility) parameters and DNA status assessed both as DNA fragmentation index (DFI) and by TUNEL assay using sperm of 105 Holstein–Friesian bulls (four batches per bull) selected based on in vivo estimated relative conception rates (ERCR). Thereafter, sperm quality and male fertility traits of bulls were explored by GWAS using a high‐density (777K) Illumina chip. After data editing, 85 bulls and 591,988 SNPs were retained for GWAS. Of 12 SNPs with false discovery rate <0.2, four SNPs located on BTA28 and BTA18 were significantly associated (LD‐adjusted Bonferroni <0.05) with the non‐compensatory sperm parameters DFI and TUNEL. Other SNPs of interest for potential association with TUNEL were found on BTA3, in the same chromosome where associations with non‐compensatory in vivo bull fertility were already reported. Further suggestive SNPs for sperm membrane integrity were located on BTA28, the chromosome where QTL studies previously reported associations with sperm quality traits. Suggestive SNPs for ERCR were found on BTA18 in the vicinity of a site already associated with in vivo bull fertility. Additional SNPs associated with ERCR and sperm kinetic parameters were also identified. In contrast to other, but very few GWAS on fertility traits in bovine spermatozoa, which reported significant SNPs located on BTX, we have not identified SNPs of interest in this sexual chromosome.     

Interrelationships Between Apoptosis and Fertility in Bull Sperm

Male fertility, the ability of sperm to fertilize and activate the egg and support early embryogenesis, is vital for mammalian reproduction. Despite producing adequate numbers of sperm with normal motility and morphology, some males suffer from low fertility whose molecular mechanisms are not known. The objective was to determine apoptosis in sperm from high and low fertility bulls and its relationship with male fertility. DNA damage, phosphatidylserine (PS) translocation, and expression of pro- and anti-apoptotic proteins (BAX and BCL-2) in the sperm were determined using TUNEL, Annexin V, and immunoblotting approaches, respectively. Amounts of apoptotic spermatozoa were 2.86 (± 1.31) and 3.00 (± 0.96) in high and low fertility bulls, respectively (P=0.548), and were not correlated with fertility. There was a negative correlation between early necrotic spermatozoa and viable spermatozoa (r = –0.99, P<0.0001). Fertility scores were correlated with live spermatozoa detected by eosin-nigrosin test and necrotic spermatozoa determined via flow cytometry (r = –0.49, P<0.006 and r = –0.266, P<0.0113, respectively). BAX level was higher in low fertile group than high fertile group; however, this difference was not statistically significant due to the variations of bull samples (Bull 1–3 vs. Bull 4–5) in low fertile group (P<0.283). BCL-2 was not detectable in any of the sperm samples. The results shed light onto molecular and cellular underpinnings of male fertility.     

The Relationship Between Post‐Thaw Sperm DNA Integrity and Non‐Return Rate Among Norwegian Cross‐Bred Rams

With the aim of investigating the relationship between sperm DNA integrity and non‐return rate (NRR) among Norwegian cross‐bred rams, semen from 15 individuals was examined by flow cytometry. Sperm Chromatin Structure Assay (SCSA) quantifies the proportion of spermatozoa with denatured DNA after in situ acid treatment, and the four parameters % DFI, % HDS, MEAN DFI and SD DFI are all different measures of DNA denaturation and maturation. Field fertility, reported as NRR 25 days after insemination was based on all inseminations from a large‐scale breeding programme and supplied by the Norwegian Association of Sheep and Goat Farmers. From each ram, four straws from four different weeks of the breeding season were analysed, and the associations between 25‐day NRR and the mean of the four SCSA parameters were tested using a logistic regression model. The results revealed no association between fertility and % DFI or % HDS, while SD DFI and MEAN DFI showed a significant negative association with NRR. Further, the SCSA values varied significantly between ejaculates within ram among some of the rams in the study. However, no significant association was seen between these intra‐individual differences in sperm DNA integrity and NRR. In conclusion, this study suggests an association between sperm DNA integrity and NRR for rams. However, further research must be conducted to confirm these findings and determine whether sperm DNA assessments can be applied to predict ram fertility.